Cytomegalovirus is a genus of virus that belongs to the viral family known as Herpesviridae or herpesviruses. The species that infects humans is commonly known as human cytomegalovirus (HCMV) or human herpesvirus-5 (HHV-5). Within Herpesviridae, HCMV belongs to the Betaherpesvirinae subfamily, which also includes cytomegaloviruses from other mammals.
Although they may be found throughout the body, HCMV infections are frequently associated with the salivary glands. HCMV infects between 50% and 80% of adults in the United States (40% worldwide), as indicated by the presence of antibodies in much of the general population. HCMV infection is typically unnoticed in healthy people, but can be life-threatening for the immunocompromised, such as HIV-infected persons, organ transplant recipients, or new born infants. HCMV is the virus most frequently transmitted to a developing fetus. After infection, HCMV has an ability to remain latent within the body for the lifetime of the host, with occasional reactivations from latency. Given the severity and importance of this disease, obtaining an effective vaccine is considered a public health top priority (Sung, H., et al., (2010) Expert review of vaccines 9, 1303-1314; Schleiss, Expert Opin Ther Pat. April 2010; 20(4): 597-602).
The genomes of over 20 different HCMV strains have been sequenced, including those of both laboratory strains and clinical isolates. For example, the following strains of HCMV have been sequenced: Towne (GL239909366), AD169 (GI:219879600), Toledo (GL290564358) and Merlin (GI: 155573956). HCMV strains AD169, Towne and Merlin can be obtained from the American Type Culture Collection (ATCC VR538, ATCC VR977 and ATCC VR1590, respectively).
CMV contains an unknown number of membrane protein complexes. Of the approximately 30 known glycoproteins in the viral envelope, gH and gL have emerged as particularly interesting due to their presence in several different complexes: dimeric gH/gL, trimeric gH/gL/gO (also known as the gCIII complex), and the pentameric gH/gL/pUL128/pUL130/pUL131 (pUL131 is also referred to as “pUL131A”, “pUL131a”, or “UL131A”; pUL128, pUL130, and pUL131 subunits sometimes are also referred as UL128, UL130, UL131). CMV is thought to use the pentameric complexes to enter epithelial and endothelial cells by endocytosis and low-pH-dependent fusion but it is thought to enter fibroblasts by direct fusion at the plasma membrane in a process involving gH/gL or possibly gH/gL/gO. The gH/gL and/or gH/gL/gO complex(es) is/are sufficient for fibroblast infection, whereas the pentameric complex is required to infect endothelial and epithelial cells.
The pentameric complex is considered as a major target for CMV vaccination. Viral genes UL128, UL130 and UL131 are needed for endothelial entry (Hahn, Journal of Virology 2004; 78:10023-33). Fibroblast-adapted non-endothelial tropic strains contain mutations in at least one of these three genes. Towne strain, for example, contains a 2 base pair insertion causing a frame shift in UL130 gene, whereas AD169 contains a 1 base pair insertion in UL131 gene. Both Towne and AD169 could be adapted for growth in endothelial cells, and in both instances, the frame shift mutations in UL130 or UL131 genes were repaired.
U.S. Pat. No. 7,704,510 discloses that pUL131A is required for epithelial cell tropism. U.S. Pat. No. 7,704,510 also discloses that pUL128 and pUL130 form a complex with gH/gL, which is incorporated into virions. This complex is required to infect endothelial and epithelial cells but not fibroblasts. Anti-CD46 antibodies were found to inhibit HCMV infection of epithelial cells.
CMV vaccines tested in clinical trials include Towne vaccine, Towne-Toledo chimeras, an alpha virus replicon with gB as the antigen, gB/MF59 vaccine, a gB vaccine produced by GlaxoSmithKline, and a DNA vaccine using gB and pp65. pp65 is viral protein that is a potent inducer of CD8+ responses directed against CMV. These vaccines are all poor inducers of antibodies that block viral entry into endothelial/epithelial cells (Adler, S. P. (2013), British Medical Bulletin, 107, 57-68. doi:10.1093/bmb/Idt023).
It is generally believed that neutralizing antibodies against the pentameric complex (gH/gLpUL128/pUL130/pUL131) will be significantly more potent than neutralizing antibodies raised against CMV gB subunit, or gH/gL dimeric complex. Therefore, to develop an effective vaccine against CMV, there is an urgent need for production of large quantities (e.g., commercial scales) of CMV pentameric complex.
However, recombinant production of CMV pentameric complex remains challenging. All five subunits need to be expressed (preferably at a substantially equal amount, and preferably at a sustained period of time), fold correctly, and be properly assembled into a pentamer. In addition, there is also a need to avoid undesired assembly of contaminating complexes (such as gH/gL dimer, gH/gL tetramer (two copies of gH and two copies of gL), gH/gLpUL128/pUL130 tetramer, etc.).
Recombinant expression such a eukaryotic protein complex would require the identification of suitable constructs and suitable hosts, for the expression of sufficient amounts of properly folded proteins at a sustained period of time, which can then be properly assembled into protein complexes. Furthermore, the selection of a suitable expression host has a significant impact on protein yield and quality, as well as on actual costs of the production process.